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Study Question: Can genome-wide genotyping data be analysed using a hypothesis-driven approach to enhance the understanding of the genetic basis of severe spermatogenic failure (SPGF) in male infertility?, Summary Answer: Our findings revealed a significant association between SPGF and the SHOC1 gene and identified three novel genes ( PCSK4 , AP3B1 , and DLK1 ) along with 32 potentially pathogenic rare variants in 30 genes that contribute to this condition., What Is Known Already: SPGF is a major cause of male infertility, often with an unknown aetiology. SPGF can be due to either multifactorial causes, including both common genetic variants in multiple genes and environmental factors, or highly damaging rare variants. Next-generation sequencing methods are useful for identifying rare mutations that explain monogenic forms of SPGF. Genome-wide association studies (GWASs) have become essential approaches for deciphering the intricate genetic landscape of complex diseases, offering a cost-effective and rapid means to genotype millions of genetic variants. Novel methods have demonstrated that GWAS datasets can be used to infer rare coding variants that are causal for male infertility phenotypes. However, this approach has not been previously applied to characterize the genetic component of a whole case-control cohort., Study Design Size Duration: We employed a hypothesis-driven approach focusing on all genetic variation identified, using a GWAS platform and subsequent genotype imputation, encompassing over 20 million polymorphisms and a total of 1571 SPGF patients and 2431 controls. Both common (minor allele frequency, MAF > 0.01) and rare (MAF < 0.01) variants were investigated within a total of 1797 loci with a reported role in spermatogenesis. This gene panel was meticulously assembled through comprehensive searches in the literature and various databases focused on male infertility genetics., Participants/materials Setting Methods: This study involved a European cohort using previously and newly generated data. Our analysis consisted of three independent methods: (i) variant-wise association analyses using logistic regression models, (ii) gene-wise association analyses using combined multivariate and collapsing burden tests, and (iii) identification and characterisation of highly damaging rare coding variants showing homozygosity only in SPGF patients., Main Results and the Role of Chance: The variant-wise analyses revealed an association between SPGF and SHOC1 -rs12347237 ( P  =   4.15E-06, odds ratio = 2.66), which was likely explained by an altered binding affinity of key transcription factors in regulatory regions and the disruptive effect of coding variants within the gene. Three additional genes ( PCSK4 , AP3B1 , and DLK1 ) were identified as novel relevant players in human male infertility using the gene-wise burden test approach ( P  <   5.56E-04). Furthermore, we linked a total of 32 potentially pathogenic and recessive coding variants of the selected genes to 35 different cases., Large Scale Data: Publicly available via GWAS catalog (accession number: GCST90239721)., Limitations Reasons for Caution: The analysis of low-frequency variants presents challenges in achieving sufficient statistical power to detect genetic associations. Consequently, independent studies with larger sample sizes are essential to replicate our results. Additionally, the specific roles of the identified variants in the pathogenic mechanisms of SPGF should be assessed through functional experiments., Wider Implications of the Findings: Our findings highlight the benefit of using GWAS genotyping to screen for both common and rare variants potentially implicated in idiopathic cases of SPGF, whether due to complex or monogenic causes. The discovery of novel genetic risk factors for SPGF and the elucidation of the underlying genetic causes provide new perspectives for personalized medicine and reproductive counselling., Study Funding/competing Interests: This work was supported by the Spanish Ministry of Science and Innovation through the Spanish National Plan for Scientific and Technical Research and Innovation (PID2020-120157RB-I00) and the Andalusian Government through the research projects of 'Plan Andaluz de Investigación, Desarrollo e Innovación (PAIDI 2020)' (ref. PY20&#95;00212) and 'Proyectos de Investigación aplicada FEDER-UGR 2023' (ref. C-CTS-273-UGR23). S.G.-M. was funded by the previously mentioned projects (ref. PY20&#95;00212 and PID2020-120157RB-I00). A.G.-J. was funded by MCIN/AEI/10.13039/501100011033 and FSE 'El FSE invierte en tu futuro' (grant ref. FPU20/02926). IPATIMUP integrates the i3S Research Unit, which is partially supported by the Portuguese Foundation for Science and Technology (FCT), financed by the European Social Funds (COMPETE-FEDER) and National Funds (projects PEstC/SAU/LA0003/2013 and POCI-01-0145-FEDER-007274). S.S. is supported by FCT funds (10.54499/DL57/2016/CP1363/CT0019), ToxOmics-Centre for Toxicogenomics and Human Health, Genetics, Oncology and Human Toxicology, and is also partially supported by the Portuguese Foundation for Science and Technology (UIDP/00009/2020 and UIDB/00009/2020). S. Larriba received support from Instituto de Salud Carlos III (grant: DTS18/00101), co-funded by FEDER funds/European Regional Development Fund (ERDF)-a way to build Europe) and from 'Generalitat de Catalunya' (grant 2021SGR052). S. Larriba is also sponsored by the 'Researchers Consolidation Program' from the SNS-Dpt. Salut Generalitat de Catalunya (Exp. CES09/020). All authors declare no conflict of interest related to this study., Competing Interests: The authors declare that the research was conducted without any conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

Selenocysteine, the 21st amino acid specified by the genetic code, is a rare selenium-containing residue found in the catalytic site of selenoprotein oxidoreductases. Selenocysteine is analogous to the common cysteine amino acid, but its selenium atom offers physical-chemical properties not provided by the corresponding sulfur atom in cysteine. Catalytic sites with selenocysteine in selenoproteins of vertebrates are under strong purifying selection, but one enzyme, glutathione peroxidase 6 (GPX6), independently exchanged selenocysteine for cysteine <100 million years ago in several mammalian lineages. We reconstructed and assayed these ancient enzymes before and after selenocysteine was lost and up to today and found them to have lost their classic ability to reduce hydroperoxides using glutathione. This loss of function, however, was accompanied by additional amino acid changes in the catalytic domain, with protein sites concertedly changing under positive selection across distant lineages abandoning selenocysteine in glutathione peroxidase 6. This demonstrates a narrow evolutionary range in maintaining fitness when sulfur in cysteine impairs the catalytic activity of this protein, with pleiotropy and epistasis likely driving the observed convergent evolution. We propose that the mutations shared across distinct lineages may trigger enzymatic properties beyond those in classic glutathione peroxidases, rather than simply recovering catalytic rate. These findings are an unusual example of adaptive convergence across mammalian selenoproteins, with the evolutionary signatures possibly representing the evolution of novel oxidoreductase functions., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Selenium (Se), a sparse element on earth, is an essential micronutrient in the vertebrate diet and its intake depends on its content in soils and waters worldwide. Selenium is required due to its function in selenoproteins, which contain selenocysteine (Sec), the 21st amino acid in the genetic code, as one of their constituent residues. Selenocysteine is analogous to the amino acid cysteine (Cys), which uses the abounding element sulfur instead. Despite the irregular distribution of Se worldwide, its distinct biochemical properties have made the substitution of Sec for Cys rare in vertebrate proteins. Still, vertebrates inhabited environments with different amounts of Se and may have distinctly adapted to it. To address this question, we compared the evolutionary forces acting on the coding sequences of selenoprotein genes and genes that regulate Se between vertebrate clades and between the Se-dependent genes and their paralogs with Cys. We find that the strength of natural selection in genes that use or regulate Se is distinct between land vertebrates and teleost fishes and more variable than in the Cys paralogs, particularly in genes involved in the preferential supply of Se to some organs and the tissue-specific expression of selenoproteins. This is compatible with vertebrates adapting to Se scarcity in land and its abundance in waters. In agreement, teleost fishes duplicated and subfunctionalized or neofunctionalized selenoprotein genes and maintained their capacity for Se transport in the body, which declined (under neutrality) for millions of years in terrestrial vertebrates. Dietary Se has thus distinctly shaped vertebrate evolution.

As humans migrated around the world, they came to inhabit environments that differ widely in the soil levels of certain micronutrients, including selenium (Se). Coupled with cultural variation in dietary practices, these migrations have led to a wide range of Se intake levels in populations around the world. Both excess and deficiency of Se in the diet can have adverse health consequences in humans, with severe Se deficiency resulting in diseases of the bone and heart. Se is required by humans mainly due to its function in selenoproteins, which contain the amino acid selenocysteine as one of their constituent residues. To understand the evolution of the use of this micronutrient in humans, we surveyed the patterns of polymorphism in all selenoprotein genes and genes involved in their regulation in 50 human populations. We find that single nucleotide polymorphisms from populations in Asia, particularly in populations living in the extreme Se-deficient regions of China, have experienced concerted shifts in their allele frequencies. Such differentiation in allele frequencies across genes is not observed in other regions of the world and is not expected under neutral evolution, being better explained by the action of recent positive selection. Thus, recent changes in the use and regulation of Se may harbor the genetic adaptations that helped humans inhabit environments that do not provide adequate levels of Se in the diet., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

SelenoDB (http://www.selenodb.org) aims to provide high-quality annotations of selenoprotein genes, proteins and SECIS elements. Selenoproteins are proteins that contain the amino acid selenocysteine (Sec) and the first release of the database included annotations for eight species. Since the release of SelenoDB 1.0 many new animal genomes have been sequenced. The annotations of selenoproteins in new genomes usually contain many errors in major databases. For this reason, we have now fully annotated selenoprotein genes in 58 animal genomes. We provide manually curated annotations for human selenoproteins, whereas we use an automatic annotation pipeline to annotate selenoprotein genes in other animal genomes. In addition, we annotate the homologous genes containing cysteine (Cys) instead of Sec. Finally, we have surveyed genetic variation in the annotated genes in humans. We use exon capture and resequencing approaches to identify single-nucleotide polymorphisms in more than 50 human populations around the world. We thus present a detailed view of the genetic divergence of Sec- and Cys-containing genes in animals and their diversity in humans. The addition of these datasets into the second release of the database provides a valuable resource for addressing medical and evolutionary questions in selenium biology.

Objectives: In this study we investigated the in vitro fungistatic and fungicidal activities of CPA18 and CPA109, two azole compounds with original structural features, alone and in combination with fluconazole against fluconazole-susceptible and -resistant Candida albicans strains., Methods: Antifungal activities were measured by MIC evaluation and time-kill studies. Azole binding analysis was performed by UV-Vis spectroscopy. Hyphal growth inhibition and filipin and propidium iodide staining assays were used for morphological analysis. An analysis of membrane lipids was also performed to gauge alterations in membrane composition and integrity. Synergism was calculated using fractional inhibitory concentration indices (FICIs). Evaluation of cytotoxicity towards murine macrophages was performed to verify selective antifungal activity., Results: Even though their binding affinity to C. albicans Erg11p is comparable to that of fluconazole, CPA compounds are active against resistant strains of C. albicans with a mutation in ERG11 sequences and/or overexpressing the ABC transporter genes CDR1 and CDR2, which encode ATP-dependent efflux pumps. Moreover, CPA18 is fungistatic, even against the two resistant strains, and was found to be synergistic with fluconazole. Differently from fluconazole and other related azoles, CPA compounds induced marked changes in membrane permeability and dramatic alterations in membrane lipid composition., Conclusions: Our outcomes suggest that CPA compounds are able to overcome major mechanisms of resistance in C. albicans. Also, they are promising candidates for combination treatment that could reduce the toxicity caused by high fluconazole doses, particularly in immunocompromised patients.

Selenocysteine (Sec), the 21st amino acid, is incorporated into proteins through the recoding of a termination codon, an inefficient translational process mediated by a complex molecular machinery. Sec is a rare amino acid in extant proteins, chemically similar to cysteine (Cys), found in homologous position to Cys of nonselenoprotein families. Selenoproteins account for the dependence of vertebrates on environmental selenium (Se) and have an important role in several Se-deficiency diseases. Selenoproteins are poorly characterized enzymes and reports on the functional exchangeability of Sec with Cys are limited and controversial. Whether the unique role of Sec in some selenoenzymes illustrates the broader contribution of Se to protein function is unknown (Gromer S, Johansson L, Bauer H, Arscott LD, Rauch S, Ballou DP, Williams CH Jr, Schirmer RH, Arnér ES. 2003. Active sites of thioredoxin reductases: why selenoproteins? Proc Natl Acad Sci USA. 100:12618-12623). Here, we address this question from an evolutionary perspective by the simultaneous identification of the patterns of divergence in almost half a billion years of vertebrate evolution and diversity within the human lineage for the full complement of enzymatic Sec residues in these proteomes. We complete this analysis with data for the homologous Cys residues in the same genomes. Our results indicate concerted purifying selection across Sec and Cys sites in all selenoproteomes, consistent with a unique role of Sec in protein function, low exchangeability, and an unknown degree of functional divergence with Cys homologs. The distinct biochemical properties of Sec, rather than the geographical distribution of Se, global O(2) levels or Sec metabolic cost, appear to play a major role in driving adaptive changes in vertebrate selenoproteomes. A better understanding of the selenoproteomes and neutral evolutionary patterns in other taxa will be necessary to fully assess the generality of this conclusion.

Selenoproteins are a diverse group of proteins usually misidentified and misannotated in sequence databases. The presence of an in-frame UGA (stop) codon in the coding sequence of selenoprotein genes precludes their identification and correct annotation. The in-frame UGA codons are recoded to cotranslationally incorporate selenocysteine, a rare selenium-containing amino acid. The development of ad hoc experimental and, more recently, computational approaches have allowed the efficient identification and characterization of the selenoproteomes of a growing number of species. Today, dozens of selenoprotein families have been described and more are being discovered in recently sequenced species, but the correct genomic annotation is not available for the majority of these genes. SelenoDB is a long-term project that aims to provide, through the collaborative effort of experimental and computational researchers, automatic and manually curated annotations of selenoprotein genes, proteins and SECIS elements. Version 1.0 of the database includes an initial set of eukaryotic genomic annotations, with special emphasis on the human selenoproteome, for immediate inspection by selenium researchers or incorporation into more general databases. SelenoDB is freely available at http://www.selenodb.org.

Selenocysteine (Sec) is co-translationally inserted into selenoproteins in response to codon UGA with the help of the selenocysteine insertion sequence (SECIS) element. The number of selenoproteins in animals varies, with humans having 25 and mice having 24 selenoproteins. To date, however, only one selenoprotein, thioredoxin reductase, has been detected in Caenorhabditis elegans, and this enzyme contains only one Sec. Here, we characterize the selenoproteomes of C.elegans and Caenorhabditis briggsae with three independent algorithms, one searching for pairs of homologous nematode SECIS elements, another searching for Cys- or Sec-containing homologs of potential nematode selenoprotein genes and the third identifying Sec-containing homologs of annotated nematode proteins. These methods suggest that thioredoxin reductase is the only Sec-containing protein in the C.elegans and C.briggsae genomes. In contrast, we identified additional selenoproteins in other nematodes. Assuming that Sec insertion mechanisms are conserved between nematodes and other eukaryotes, the data suggest that nematode selenoproteomes were reduced during evolution, and that in an extreme reduction case Sec insertion systems probably decode only a single UGA codon in C.elegans and C.briggsae genomes. In addition, all detected genes had a rare form of SECIS element containing a guanosine in place of a conserved adenosine present in most other SECIS structures, suggesting that in organisms with small selenoproteomes SECIS elements may change rapidly.

A publication by Raufaste and Rousset (2001) questioned the effectiveness of the partial Mantel test, a nonparametric statistical test for association among three distance matrices. By repeated simulations, we calculated the cumulative density functions of the null-hypothesis probability of no correlation, within an explicit model of causal relationships. Results do not support the criticism: in conditions of moderate correlation between the independent matrices, the actual error rate is closely associated to the intended type-I error alpha.

The poly(ADP-ribose) polymerase-like thermozyme purified from Sulfolobus solfataricus was characterised with respect to some physico-chemical properties. The archaeal protein exhibited a scarce electrophoretic mobility at both pH 2.9 and pH 7.5. Determination of the isoelectric point (pI=7.0-7.2) allowed us to understand the reason for the limited migration at pH 7.5, while amino acid composition analysis showed a moderate content of basic residues, which reduced mobility at pH 2.9. With respect to the charge, the archaeal enzyme behaved differently from the eukaryotic thermolabile poly(ADP-ribose) polymerase, described as a basic protein (pI=9.5). Well known inhibitors of the mesophilic polymerase like Zn(2+), nicotinamide and 3-aminobenzamide exerted a smaller effect on the enzyme from S. solfataricus, reducing the activity by at most 50%. Mg(2+) was a positive effector, although in a dose-dependent manner. It influenced the fluorescence spectrum of the archaeal protein, whereas NaCl had no effect.